RNA respiratory syncytial virus vaccines

ABSTRACT

A vector comprising a first DNA sequence which is complementary to at least part of an alphavirus RNA genome and having the complement of complete alphavirus DNA genome replication regions, a second DNA sequence encoding a paramyxovirus protein, particularly a respiratory syncytial virus fusion (RSV F) protein or a RSV F protein fragment that generates antibodies that specifically react with RSV F protein, the first and second DNA sequences being under the transcriptional control of a promoter is described. Such vector may be used to produce an RNA transcript which may be used to immunize a host, including a human host, to protect the host against disease caused by paramyxovirus, particularly respiratory syncytial virus, by administration to the host. The RNA transcript may be formed by linearization of the vector through cleavage at a unique restriction site in a plasmid vector and then transcribing the linear molecule.

This application is a U.S. National Phase filing pursuant to 35 U.S.C371 of PCT/CA98/00840 filed Sep. 3, 1998 which itself is acontinuation-in-part of U.S. patent application Ser. No. 08/923,558filed Sep. 4, 1997 (now U.S. Pat. No.: 6,060,308).”

FIELD OF INVENTION

The present invention relates to the field of paramyxoviridae vaccinesand is particularly concerned with vaccines comprising RNA encoding thefusion (F) protein of respiratory syncytial virus (RSV).

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (RSV) has been identified as a majorpathogen responsible for severe respiratory tract infections in infants,young children and the institutionalized elderly (refs.1,2,3,4—throughout this application, various references are cited inparentheses to describe more fully the state of the art to which thisinvention pertains. Full bibliographic information for each citation isfound at the end of the specification, immediately preceding the claims.The disclosures of these references are hereby incorporated by referenceinto the present disclosure). Global mortality and morbidity figuresindicate that there is an urgent need for an efficacious RSV vaccine(refs. 5,6). In the USA alone, approximately 100,000 children arehospitalized annually with severe cases of pneumonia and bronchiolitisresulting from an RSV infection. Inpatient and ambulatory care forchildren with RSV infections has been estimated to cost in excess of$340 million each year in the USA. The World Health Organization (WHO)and the National Institute of Allergy and Infectious Disease (NIAID)vaccine advisory committees have ranked RSV second only to HIV forvaccine development. Both the annual morbidity and mortality figures aswell as the staggering health care costs for managing RSV infectionshave provided the incentive for aggressively pursuing the development ofefficacious RSV vaccines. However, such a vaccine is still notavailable.

Formalin-inactivated (FI-RSV) and live attenuated RSV vaccines havefailed to demonstrate efficacy in clinical trials (refs. 7,8,9,10).Moreover, the formalin-inactivated RSV vaccine caused enhanced diseasein some children following exposure to wild-type RSV (refs. 7,8,9,10).Elucidation of the mechanism(s) involved in the potentiation of RSVdisease is important for the design of safe RSV vaccines, especially forthe seronegative population. Recent experimental evidence suggests thatan imbalance in cell-mediated responses may contribute toimmunopotentiation. Enhanced histopathology observed in mice that wereimmunized with the FI-RSV and challenged with virus could be abrogatedby depletion of CD4+ cells or both interleukin-4 (IL-4) and IL-10.

The RSV fusion (F) glycoprotein is one of the major immunogenic proteinsof the virus. This envelope glycoprotein mediates both fusion of thevirus to the host cell membrane and cell-to-cell spread of the virus(ref. 1). The F protein is synthesized as a precursor (F₀) moleculewhich is proteolytically cleaved to form a disulphide-linked dimercomposed of the N-terminal F₂ and C-terminal F₁ moieties (ref. 11). Theamino acid sequence of the F protein is highly conserved among RSVsubgroups A and B and is a cross-protective antigen (refs. 6,12). In thebaculovirus expression system, a truncated secreted version of the RSV Fprotein has been expressed in Trichoplusia ni insect cells (ref. 13).The recombinant protein was demonstrated to be protective in the cottonrats (ref. 13).

Studies on the development of live viral vaccines and glycoproteinsubunit vaccines against parainfluenza virus infection are beingpursued. Clinical trial results with a formalin-inactivated PIV types1,2,3 vaccine demonstrated that this vaccine was not efficacious (refs.14, 15, 16). Further development of chemically-inactivated vaccines wasdiscontinued after clinical trials with a formalin-inactivated RSVvaccine demonstrated that not only was the vaccine not effective inpreventing RSV infection but many of the vaccinees who later becameinfected with RSV suffered a more serious disease. Most of parainfluenzavaccine research has focussed on candidate PIV-3 vaccines (ref. 17) withsignificantly less work being reported for PIV-1 and PIV-2. Recentapproaches to PIV-3 vaccines have included the use of the closelyrelated bovine parainfluenza virus type 3 and the generation ofattenuated viruses by cold-adaptation of the virus (refs. 18, 19, 20,21).

Another approach to parainfluenza virus type 3 vaccine development is asubunit approach focusing on the surface glycoproteinshemagglutinin-neuraminidase (HN) and the fusion (F) protein (refs. 22,23, 24). The HN antigen, a typical type II glycoprotein, exhibits bothhaemagglutination and neuraminidase activities and is responsible forthe attachment of the virus to sialic acid containing host cellreceptors. The type I F glycoprotein mediates fusion of the viralenvelope with the cell membrane as well as cell to cell spread of thevirus. It has recently been demonstrated that both the HN and Fglycoproteins are required for membrane fusion. The F glycoprotein issynthesized as an inactive precursor (F) which is proteolyticallycleaved into disulfide-linked F2 and F1 moieties. While the HN and Fproteins of PIV-1, -2 and -3 are structurally similar, they areantigenically distinct. Neutralizing antibodies against the HN and Fproteins of one of PIV type are not cross-protective. Thus, an effectivePIV subunit vaccine must contain the HN and F glycoproteins from thethree different types of parainfluenza viruses. Antibody to eitherglycoprotein is neutralizing in vitro. A direct correlation has beenobserved between the level of neutralizing antibody titres andresistance to PIV-3 infections in infants. Native subunit vaccines forparainfluenza virus type 3 have investigated the protectiveness of thetwo surface glycoproteins. Typically, the glycoproteins are extractedfrom virus using non-ionic detergents and further purified using lectinaffinity or immunoaffinity chromatographic methods. However, neither ofthese techniques may be entirely suitable for large scale production ofvaccines under all circumstances. In small animal protection models(hamsters and cotton rats), immunization with the glycoproteins wasdemonstrated to prevent infection with live PIV-3 (refs. 25, 26, 27, 28,29). The HN and F glycoproteins of PIV-3 have also been produced usingrecombinant DNA technology. HN and F glycoproteins have been produced ininsect cells using the baculovirus expression system and by use ofvaccinia virus and adenovirus recombinants (refs. 30, 31, 32, 33, 34).In the baculovirus expression system, both full-length and truncatedforms of the PIV-3 glycoproteins as well as a chimeric F-HN fusionprotein have been expressed. The in recombinant proteins have beendemonstrated to be protective in small animal models (see W091/00104,U.S. application Ser. No. 07/773,949 filed Nov. 29, 1991, assigned tothe assignee hereof).

Semliki Forest virus (SFV) is a member of the Alphavirus genus in theTogaviridae family. The mature virus particle contains a single copy ofa ssRNA genome with a positive polarity that is 5′-capped and3′-polyadenylated. It functions as an mRNA and naked RNA can start aninfection when introduced into cells. Upon infection/transfection, the5′ two-thirds of the genome is translated into a polyprotein that isprocessed into the four nonstructural proteins (nsP1 to 4) by selfcleavage. Once the ns proteins have been synthesized they areresponsible for replicating the plus-strand (42S) genome intofull-length minus strands (ref. 35). These minus-strands then serve astemplates for the synthesis of new plus-strand (42S) genomes and the 26Ssubgenomic mRNA (ref. 35). This subgenomic mRNA, which is colinear withthe last one-third of the genome, encodes the SFV structural proteins.In 1991 Liljestrom and Garoff (ref. 36) designed a series of expressionvectors based on the SFV cDNA replicon. These alphavirus vectors alsoare described in WO 92/10578, the disclosure of which is incorporatedherein by reference. These vectors had the virus structural proteingenes deleted to make the way for heterologous inserts, but preservedthe nonstructural coding region for production of the nsP1 to 4replicase complex. Short 5′ and 3′ sequence elements required for RNAreplication were also preserved. A polylinker site was inserteddownstream from the 26S promoter followed by translation stop sites inall three frames. An SpeI site was inserted just after the 3′ end of theSFV cDNA for linearization of the plasmid for use in vitro transcriptionreactions.

Injections of SFV RNA encoding a heterologous protein have been shown toresult in the expression of the foreign protein and the induction ofantibody in a number of studies (refs. 37, 38). The use of SFV RNAinoculation to express foreign proteins for the purpose of immunizationwould have several of the advantages associated with plasmid DNAimmunization. For example, SFV RNA encoding a viral antigen may beintroduced in the presence of antibody to that virus without a loss inpotency due to neutralization by antibodies to the virus. Also, becausethe protein is expressed in vivo the protein should have the sameconformation as the protein expressed by the virus itself. Therefore,concerns about conformational changes which could occur during proteinpurification leading to a loss in immunogenecity, protective epitopesand possibly immunopotentiation, could be avoided by nucleic acidimmunization.

Immunization with SFV RNA also has several unique advantages overplasmid DNA immunization. SFV is one of the most efficiently replicatingviruses known. After a few hours, up to 200,000 copies of the plus-RNAscan be made in a single cell. These SFV RNAs are so abundant almost allof the cells ribosomes are enrolled in the synthesis of the SFV encodedproteins, thus overtaking host cell protein synthesis (ref. 36).Therefore, it should require a smaller dose of SFV RNA and less time toachieve a protective effect as compared to plasmid DNA immunization.Secondly, RNA, unlike DNA, poses no potential threat of integrating intothe cell genome. Thirdly, SFV RNA replication and expression occurs onlyin the cytoplasm of the cell. Therefore, problems involving nucleartransport and splicing associated with nucleus-based expression systems(DNA immunization) are absent. Fourthly, since the replication of theSFV RNA is transient and RNA is quite labile, the SFV RNA will notpersist for long periods after immunization like DNA plasmids.

In WO 95/27044, the disclosure of which is incorporated herein byreference, there is described the use of alphavirus cDNA vectors basedon cDNA complementary to the alphavirus RNA sequence. Once transcribedfrom the cDNA under transcriptional control of a heterlogous promoter,the alphavirus RNA is able to self-replicate by means of its ownreplicase and thereby amplify the copy number of the transcribedrecombinant RNA molecules.

In copending U.S. patent application Ser. No. 08/476,397 filed Jun. 7,1995, now U. S. Pat. No. 6,019,980 (WO 96/40945), assigned to theassignee hereof and the disclosure of which is incorporated herein byreference, there are described certain plasmid constructs used for DNAimmunization which include forms of the RSV F gene. As seen therein, oneplasmid pXL2 conferred complete protection on mice to challenge by liveRSV when administered intranasally. This plasmid contains a geneencoding a truncated RSV F protein lacking the transmembrane portion ofthe protein, the immediate-early promoter enhancer and intron sequencesof human cytomegatrovius (CMV) and the intron II sequences of rabbitβ-globin to prevent aberrant splicing. The same plasmid construct butwithout the intron II sequences of rabbit β-globin, i.e. pXL1, providedonly partial protection. Similarly, plasmid construct pXL4, which is thesame as pXL2 except the RSV F gene encodes the full length RSV protein,provided partial protection while the corresponding construct lackingthe intron II sequence of rabbit β-globin, i.e. pXL3, conferred noprotection.

These data show that the absence of elements to reduce aberrant splicingadversely affects the, protective ability of the plasmid. Aberrantsplicing occurs during nuclear transcription of DNA to RNA. By employingRNA transcripts for immunization, the need for nuclear processing isavoided and aberrant splicing is unable to occur. This enables the useof the intron II sequences from non-human sources to be avoided.

The use of RNA transcripts for administration to the host enables thereto be obtained total protection to challenge using a lower dose in lesstime than when employing the DNA plasmids described in U.S. Ser. No.08/476,397 (WO 96/40945). The use of the RNA transcripts avoidspersistence of DNA in the immunized host and potential integration.

The ability to immunize against disease caused by RSV by immunizationwith naked SFV RNA encoding the RSV F protein, particularly the secretedversion of the RSV F protein, was unknown before the present inventionand could not be predicted on the basis of the known prior art.Infection with RSV leads to serious disease. It would be useful anddesirable to provide improved vectors for in vivo administration ofimmunogenic preparations, including vaccines, for protection againstdisease caused by RSV. In particular, it would be desirable to providevaccines that are immunogenic and protective in the elderly andpaediatric human populations, including seronegative infants, that donot cause disease enhancement (immunopotentiation).

SUMMARY OF THE INVENTION

The present invention provides novel immunogenic materials andimmunization procedures based on such novel materials for immunizingagainst disease caused by paramyxoviridae, including respiratorysyncytial virus and parainfluenza virus. In particular, the presentinvention is directed towards the provision of RNA vaccines againstdisease caused by infection with paramyxoviridae.

In accordance with one aspect of the present invention, there isprovided a vector, comprising a first DNA sequence which iscomplementary to at least part of an alphavirus RNA genome and havingthe complement of complete alphavirus RNA genome replication regions; asecond DNA sequence encoding a paramyxovirus protein or a proteinfragment that generates antibodies that specifically react with theparamyxovirus protein; the second DNA sequence being inserted into aregion of the first DNA sequence which is non-essential for replication;the first and second DNA sequences being under transcriptional controlof a promoter.

The paramyxovirus protein may be selected from the group consisting of aparainfluenza virus (PIV) and a respiratory syncytial virus (RSV). ThePIV protein may be PIV-1, PIV-2, PIV-3 or PIV-4, particularly the HN orF glycoproteins of PIV-3. The RSV protein particularly may be the F or Gglycoprotein of RSV.

The second DNA sequence may encode a full length RSV F protein, or mayencode a RSV F protein lacking the transmembrane anchor and cytoplasmictail. The lack of the coding region for the transmembrane anchor andcytoplasmic tail results in a secreted form of the RSV F protein.

The second DNA sequence preferably encodes a RSV F protein and lacks aSpeI restriction site, and optionally, also lacking the transmembraneanchor and cytoplasmic tail encoding region. The absence of the SpeIrestriction site may be carried out by mutating nucleotide 194 (T) ofthe RSV F gene to a C, which eliminates the SpeI without altering theamino acid sequence. The nucleotide sequence (SEQ ID No: 1) and encodedamino acid sequence (SEQ ID No: 2) of the mutated truncated RSV F geneis shown in FIG. 2.

The alphavirus preferably is a Semliki Forest virus and the first DNAsequence is the Semliki Forest viral sequence contained in plasmidpSFV1. The promoter used preferably is the SP6 promoter.

The vector may contain a unique restriction site permittinglinearization of the vector without cleaving the second nucleotidesequence and maintaining the first and second nucleotides sequencesunder transcriptional control of the promoter. The unique restrictionsite preferably is a SpeI site, particularly that derived from pSFV1.The linearized form of the vector forms an embodiment herein.

The vector may be a plasmid vector, preferably one having theidentifying characteristics of plasmid pMP37 (ATCC 97905) as shown inFIG. 1C and, more preferably, is the plasmid pMP37.

The mutant DNA sequence encoding an RSV F protein or a fragment thereofcapable of inducing antibodies that specifically react with RSV Fprotein and lacking the SpeI restriction site present in the native DNAsequence, constitutes another aspect of the present invention. Suchmutant DNA sequence lacking the SpeI site of the native sequencepreferably is that shown in FIG. 2 (SEQ ID No: 1) or one which encodesthe amino acid sequence shown in FIG. 2 (SEQ ID No: 2).

The novel vector provided herein may be linearized by cleavage at theunique restriction site and transcribed to an RNA transcript. Inaccordance with a further aspect of the invention, there is provided anRNA transcript of a vector as provided herein produced by linearizationand transcription.

The RNA transcripts provided herein may be provided in the form of animmunogenic composition for in vivo administration to a host for thegeneration in the host of antibodies to paramyxovirus protein, suchimmunogenic compositions comprising, as the active component thereof, anRNA transcript as provided herein. Such immunogenic compositions, whichare provided in accordance with another aspect of the invention, may beformulated with any suitable pharmaceutically-acceptable carrier for thein vivo administration and may produce a protective immune response.

In a yet further aspect of the present invention, there is provided amethod of immunizing a host against disease caused by infection withparamyxovirus, which comprises administering to the host an effectiveamount of an RNA transcript as provided herein.

The present invention also includes a novel method of using a geneencoding a RSV F protein or an fragment of an RSV F protein capable ofgenerating antibodies which specifically react with RSV F protein toprotect a host against disease caused by infection with respiratorysyncytial virus, which comprises isolating said gene; operativelylinking said gene to a DNA sequence which is complementary to at leastpart of an alphavirus RNA genome and having the complement of completealphavirus RNA genome replication regions in a region of said DNAsequence which is non-essential for replication to form a plasmid vectorwherein said gene and DNA sequence are under transcriptional control ofa promoter; linearizing the plasmid vector while maintaining said geneand DNA sequence under said transcriptional control of the promoter;forming an RNA transcript of said linearized vector; and introducingsaid RNA transcript to said host.

Linearizing the plasmid vector is effected by cleaving the plasmidvector at a unique restriction site therein at a location which permitsthe maintenance of the gene and the DNA sequence under thetranscriptional control of the promoter. The unique restriction site maybe an SpeI site, such as derived from plasmid pSFV1.

The plasmid vector employed preferably is plasmid pMP37 and thelinearizing step is effected by cleavage at the SpeI site of plasmidpMP37 (see FIG. 1C).

In addition, the present invention includes a method of producing avaccine for protection of a host against disease caused by infectionwith respiratory syncytial virus (RSV), which comprises isolating afirst DNA sequence encoding an RSV F protein from which thetransmembrane anchor and cytoplasmic tail are absent and lacking anySpeI restriction site; operatively linking said first DNA sequence to asecond DNA sequence which is complementary to at least part of analphavirus RNA genome and having the complete alphavirus genomereplication regions in a region of said second DNA sequence which isnon-essential for replication to form a plasmid vector wherein saidfirst and second DNA sequences are under transcriptional control of apromoter; linearizing the plasmid vector while maintaining said firstand second DNA sequences under said transcriptional control of thepromoter; forming a RNA transcript of said linearized vector; andformulating said RNA transcript as a vaccine for in vivo administration.

Linearizing the plasmid vector is effected by cleaving the plasmidvector at a unique restriction site therein at a location which permitsthe maintenance of the gene and the DNA sequence under thetranscriptional control of the promoter. The unique restriction site maybe an SpeI site, such as derived from plasmid pSFV1.

The plasmid vector employed preferably is plasmid pMP37 and thelinearizing step is effected by cleavage at the SpeI site of plasmidpMP37.

Advantages of the present invention include the c provision of RNAtranscripts which are useful in generating an immune response by in vivoadministration.

BRIEF DESCRIPTION OF THE FIGURES

The present invention will be further understood from the followingdescription with reference to the drawings, in which:

FIGS. 1A, 1B and 1C show a scheme for construction of plasmid pMP37 usedto generate the RSV-F RNA;

FIG. 2 shows the nucleotide sequence (SEQ ID No: 1) and deduced aminoacid sequence (SEQ ID No: 2) of a truncated RSV F gene lacking thetransmembrane anchor and cytoplasmic tail and mutated at nucleotide 194to eliminate the SpeI restriction site present in the unmutated gene;

FIG. 3, comprising panels A, B and C, shows the anti-RSV F titres insera from mice taken 4 weeks after primary immunization and 2 weeksafter boosting with the RSV F RNA. Panels A, B, and C show total IgGresponse, IgG1 response and IgG2a response respectively; and

FIG. 4 shows the RSV-specific neutralizing antibody titres expressed asplaque reduction titres for various RSV preparations.

GENERAL DESCRIPTION OF INVENTION

As described above, the present invention, in general, relates toprotection of hosts against disease caused by infection by paramyxovirusby RNA immunization using RNA transcripts formed from DNA vectors bylinearization and transcription of the linearized vector. In particular,the invention is concerned with protection of hosts against diseasecaused by infection by respiratory syncytial virus (RSV), although notspecifically limited thereto. The description which follows refersspecifically to employing DNA sequences and RNA transcripts thereofencoding RSV F protein and fragments thereof which generate antibodieswhich specifically react with RSV F protein.

In this application, the term “RSV F protein” is used to define afull-length RSV F protein, including proteins having variations in theiramino acid sequences including those naturally occurring in variousstrain of RSV and those introduced by PCR amplification of the encodinggene while retaining the immunogenic properties, a secreted form of theRSV F protein lacking a transmembrane anchor and cytoplasmic tail, aswell as fragments of RSV F protein capable of generating antibodieswhich specifically react with RSV F protein and functional analogs ofRSV F protein. In this application, a first protein is a “functionalanalog” of a second protein if the first protein is immunologicallyrelated to and/or has the same function as the second protein. Thefunctional analog may be, for example, a fragment of the protein or asubstitution, addition or deletion mutant thereof.

A vector is constructed to contain a first DNA sequence which iscomplementary to at least part of an alphavirus RNA genome, specificallySemliki Forest virus, and having the complement of complete alphavirusRNA genome replication regions. A second DNA sequence encoding the RSV Fprotein is inserted into a region of the first DNA sequence which isnon-essential for replication. The first and second DNA sequences areunder transcriptional control of a promoter. The resulting vector islinearized and an RNA transcript formed from the linearized vector.

The RNA transcripts provided herein, when administered to an animal,including a human, replicate rapidly and effect in vivo RSV F proteinexpression, as demonstrated by an RSV F-protein specific antibodyresponse in the animal to which it is administered. Such antibodies maybe employed, if desired, in the detection of RSV protein in a sample.

As may be seen from the results detailed in the Examples below, the RNAtranscripts provided a high anti-F IgG antibody titre with a IgG1/IgG2aratio closely following the ratio obtained from immunization with livevirus. Immunization with the RNA transcripts protected the animalsagainst live RSV challenge.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis and treatment of RSV infections. Afurther non-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the RSV F gene and vectors as disclosed herein. Thevaccine elicits an immune response in a subject which includes theproduction of anti-RSV F antibodies. Immunogenic compositions, includingvaccines, containing the RNA transcripts may be prepared as injectables,in physiologically-acceptable liquid solutions or emulsions forpolynucleotide administration. The RNA transcripts associated withliposomes, such as lecithin liposomes or other liposomes known in theart, as a nucleic acid liposome (for example, as described in WO93/24640, ref. 38) or the RNA may be associated with an adjuvant, asdescribed in more detail below. Liposomes comprising cationic lipidsinteract spontaneously and rapidly with polyanions, such as DNA and RNA,resulting in liposome/nucleic acid complexes that capture up to loot ofthe polynucleotide. In addition, the polycationic complexes fuse withcell membranes, resulting in an intracellular delivery of polynucleotidethat bypasses the degradative enzymes of the lyposomal compartment.Published PCT application WO 94/27435 describes compositions for geneticimmunization comprising cationic lipids and polynucleotides. Agentswhich assist in the cellular uptake of nucleic acid, such as calciumions, viral proteins and other transfection facilitating agents, mayadvantageously be used.

Polynucleotide immunogenic preparations may also be formulated asmicrocapsules, including biodegradable time-release particles. Thus,U.S. Pat. No. 5,151,264 describes a particulate carrier of aphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moleculaires (BVSM). The particulate carriers areintended to transport a variety of molecules having biological activityin one of the layers thereof.

U.S. Pat. No. 5,075,109 describes encapsulation of the antigenstrinitrophenylated keyhole limpet hemocyanin and staphylococcalenterotoxin B in 50:50 poly (DL-lactidecoglycolide). Other polymers forencapsulation are suggested, such as poly(glycolide),poly(DL-lactide-coglycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(8-hydroxybutyric acid), and polyanhydrides.

Published PCT application WO 91/06282 describes a delivery vehiclecomprising a plurality of bioadhesive microspheres and antigens. Themicrospheres being of starch, gelatin, dextran, collagen or albumin.This delivery vehicle is particularly intended for the uptake of vaccineacross the nasal mucosa. The delivery vehicle may additionally containan absorption enhancer.

The RNA transcripts may be mixed with pharmaceutically acceptableexcipients which are compatible therewith. Such excipients may includewater, saline, dextrose, glycerol, ethanol, and combinations thereof.The immunogenic compositions and vaccines may further contain auxiliarysubstances, such as wetting or emulsifying agents, pH buffering agents,or adjuvants to enhance the effectiveness thereof. Immunogeniccompositions and vaccines may be administered parenterally, by injectionsubcutaneously, intravenously, intradermally or intramuscularly,possibly following pretreatment of the injection site with a localanesthetic. Alternatively, the immunogenic compositions formed accordingto the present invention, may be formulated and delivered in a manner toevoke an immune response at mucosal surfaces. Thus, the immunogeniccomposition may be administered to mucosal surfaces by, for example, thenasal or oral (intragastric) routes. Alternatively, other modes ofadministration including suppositories and oral formulations may bedesirable. For suppositories, binders and carriers may include, forexample, polyalkalene glycols or triglycerides. Oral formulations mayinclude normally employed incipients, such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizethe RSV F protein and antibodies thereto, and if needed, to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of about 1 μg to about 10 mg of theRSV F RNA. Suitable regimes for initial administration and booster dosesare also variable, but may include an initial administration followed bysubsequent administrations. The dosage may also depend on the route ofadministration and will vary according to the size of the host. Avaccine which protects against only one pathogen is a monovalentvaccine. Vaccines which contain antigenic material of several pathogensare combined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

Immunogenicity can be significantly improved if the vectors areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Thus,adjuvants have been identified that enhance the immune response toantigens. Some of these adjuvants are toxic, however, and can causeundesirable side-effects, making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating materialcan provoke potent immune responses to antigens. These include saponinscomplexed to membrane protein antigens to produce immune stimulatingcomplexes (ISCOMS), plutonic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as monophoryl lipid A, QS 21 and polyphosphazene.

In particular embodiments of the present invention, the RNA transcriptcomprising a first nucleotide sequence encoding an F protein of RSV maybe delivered in conjunction with a targeting molecule to target thevector to selected cells including cells of the immune system.

The RNA transcript may be delivered to the host by a variety ofprocedures, for example, Tang et al. (ref. 39) disclosed thatintroduction of gold microprojectiles coated with DNA encoding bovinegrowth hormone (BGH) into the skin of mice resulted in production ofanti-BGH antibodies in the mice, while Furth et al. (ref. 40) showedthat a jet injector could be used to transfect skin, muscle, fat andmammary tissues of living animals.

Biological Deposits

Certain vectors that contain the gene encoding RSV F protein andreferred to herein have been deposited with the American Type CultureCollection (ATCC) located at 10801 University Boulevard, Manassas, Va.20110-2209, U.S.A., pursuant to the Budapest Treaty and prior to thefiling of this application.

Samples of the deposited plasmids will become available to the publicupon grant of a patent based upon this United States patent applicationand all restrictions on access to the deposits will be removed at thattime. Non-viable deposits will be replaced in the event ATCC is unableto dispense the same. The invention described and claimed herein is notto be limited in scope by plasmids deposited, since the depositedembodiment is intended only as an illustration of the invention. Anyequivalent or similar plasmids that encode similar or equivalentantigens as described in this application are within the scope of thisinvention.

Deposit Summary

Plasmid ATCC Designation Date Deposited pMP37 97905 Feb. 27, 1997

EXAMPLE

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example describes the construction of a Semliki Forest virus (SFV)expression vector containing a truncated version of the RSV F gene.

A truncated version of the RSV F gene was inserted into the SFVexpression vector pSFV1 (Gibco BRL, Gaithersburg, Md., USA) according tothe steps outlined in FIG. 1. The RSV F gene was originally cloned froma subtype A RSV clinical isolate into plasmid pRSV F, as fully describedin copending U.S. patent application Ser. No. 08/001,554 filed Jan. 6,1993, assigned to the assignee hereof and the disclosure of which isincorporated herein by reference, (ref. 41 and WO 93/14207). A fragmentof the RSV F gene was excised from plasmid RSV F by digesting theplasmid with BspHI and EcoRI. The restriction enzyme BspHI cuts withinthe RSV F gene coding region, removing 48 amino acids from theC-terminus of the F protein. These amino acids make up most of thetransmembrane domain and the entire cytoplasmic tail. The resulting 1.6Kb truncated RSV F gene fragment was cloned into the EcoRI-BamHI sitesof the Bluescript-based mammalian cell expression vector pMCR20(Stratagene, La Jolla, Calif.) in a 3-way ligation with a linker, basedupon the following sequence:

5′ CATGACTTGATAATGAG 3′ (SEQ ID No: 3)

3′ TGAACTATTACTCCTAG 5′ (SEQ ID No: 4)

to generate plasmid pES13A, as described in the aforementioned U.S. Ser.No. 08/001,554 (WO 93/14207). This linker adds a non-template encodedthreonine to the truncated RSV F protein C-terminus and inserts threesuccessive stop codons at the end of the truncated gene.

The 1.6 Kb truncated RSV F gene fragment was then excised from plasmidpES13A by digesting with EcoRI and BamHI. In another 3-way ligation, the1.6 Kb EcoRI-BamHI RSV F gene fragment was cloned into the BamHI site ofthe SFV expression vector pSFV1 with another linker, based upon thefollowing sequence:

5′ GATCCGCGCGCGCG 3′ (SEQ ID No: 5)

3′ GCGCGCGCGCTTAA 5′ (SEQ ID No: 6)

to generate plasmid pMP35. This plasmid contained two copies of the 1.6Kb BamHI RSV F gene fragment. At this time, it was discovered that therewas an SpeI site located in the RSV F gene fragment 193 bp from theupstream BamHI site. It is necessary to linearize a pSFV1 based plasmidwith SpeI prior to its use in the in vitro transcription reactiondescribed below. Therefore, the SpeI site in the RSV F gene needed to beremoved and this was effected in the following manner.

The 1.6 Kb truncated RSV F gene fragment was excised from plasmid pMP35by digesting with BamHI and ligated into the BamHI site of pUC19 togenerate the plasmid pMP36. The Transformer™ site-directed mutagenesiskit (Clonetech, Palo Alto, Calif., USA) and a primer,5′-TGGTTGGTATACCAGTGTTATAACT (SEQ ID No: 7) were used, according to themanufacturer's instructions, to change nucleotide 194 from a T to a C.This change eliminates the SpeI site in the RSV F gene without affectingthe amino acid sequence of the encoded RSV F protein. The sequence ofplasmid pMP36A, which contains the altered RSV F gene, was determined byDNA sequence analysis. The 1.6 Kb truncated RSV F gene fragment wasexcised from plasmid pMP36A by digesting with BamHI and ligated into theBamHI site of pSFV1 to generate plasmid pMP37 (ATCC 97905). Properorientation of the truncated RSV F gene was confirmed by restrictionmapping and DNA sequence analysis. FIG. 2 shows the nucleotide sequence(SEQ ID No: 1) of the truncated RSV F gene BamHI fragment with the SpeIsite eliminated and the amino acid sequence (SEQ ID No: 2) of thesecreted RSV F protein encoded thereby.

Plasmid DNA was purified using plasmid DNA mide kits from Qiagen(Chatsworth, Calif., USA), according to the manufacturer's instructions.

Example 2

This Example describes the preparation of SpeI linearized pMP37 requiredfor the generation of SFV-RSVF RNA in in vitro transcription reactionsand the preparation of SFV-RSVF RNA.

20 μg of plasmid pMP37, prepared as described in Example 1, was cut withSpeI in a 100 μL reaction containing 20 MM Tris-HCl (pH 7.4), 5 mMMgCl2, 50 mM KCl and 30 units of SpeI (Gibco BRL, Gaithersburg, Md.,USA).

SFV-RNA was generated from the linearized plasmid in a 300 μL in vitrotranscription reaction using of the following materials

40 mM Tris-HCl (pH 7.9)

6 mM MgCl₂

2 mM spermidine-(HCl)₃

1 mM DTT (dithiothreonol)

1 mM ATP (adenosine triphosphate)

1 mM GTP (guanosine triphosphate)

1 mM CTP (cytidine triphosphate)

1 mM UTP (uridine triphosphate)

1 mM m⁷G(5′)ppp(5′)G RNA cap analog (New England Biolabs, Mississauga,Ont., Canada)

360 units of RNasin® enzyme inhibitor (Promega, Madison, Wis., USA)

270 units of SP6 RNA polymerase (Gibco BRL, Gaithersburg, Md., USA)

The reaction was incubated at 37° C. for 50 minutes. The SFV-RSVF RNA soproduced was purified from the salt, enzymes, unincorporated NTP's andcap analog by passing the reaction mix through CHROMA SPIN™-200 DEPC-H₂Ocolumns (Clonetech, Palo Alto, Calif., USA) (75 μL/column) according tothe manufacturer's instructions. The purified RNA then was ethanolprecipitated and resuspended in DEPC-treated H₂O to a finalconcentration of 1 μg/μL. The purified RNA was mixed with an equalvolume of 2×PBS just prior to immunization.

Example 3

This Example describes the immunization of mice with SFV-RSVF RNA andthe immunogenicity results obtained.

It has previously been shown that mice are susceptible to infection withRSV (ref. 42) and are a relevant animal model. The mice were immunizedwith the SFV-RSVF RNA prepared as described in Example 2, by theintramuscular (i.m.) route. The anterior tibialis muscles of five BALB/cmice (female 6 to 8 week old) (Jackson Lab., Bar Harbour, Me., USA) werebilaterally injected with 2×25 μg (0.5 μg/μL) of the PBS-directedSFV-RSVF RNA. Five days prior to RNA immunization, the muscles weretreated with 2×50 μL of cardiotoxin (10 μM in PBS) (Latoxan, France).Treatment of muscles with cardiotoxin has previously been shown toenhance the uptake of DNA and enhance the immune response (ref. 43).These mice were boosted in an identical manner 4 weeks later (Table 1below). The control groups were immunized with (1) SFV RNA expressingβ-galactosidase (SFV-LacZ RNA), (2) SFV-RSVF RNA as prepared herein; (3)live RSV, (4) PBS with alum and RSV subunit preparation with alum. Thesemice were also boosted in an identical manner 4 weeks later (Table 1).The RSV subunit preparation, comprising RSV F, G and M proteins isdescribed in copending U.S. patent application Ser. No. 08/679,060 filedJul. 12, 1996 now U.S. Pat. No. 6,020,182, assigned to the assigneehereof and the disclosure of which is incorporated herein by reference(WO 98/02457).

Two weeks after the second immunization, mice were challengedintranasally with 10⁶ plaque forming units (pfu) of the A2 strain of RSV(BG-4A). Animals were sacrificed 4 days later. Lungs were ascepticallyremoved, weighed, and homogenized in 2 mL of complete culture medium.The virus titre in lung homogenates was determined in duplicate usingVero cells, as previously described (ref. 44).

Sera was obtained from the mice at 4 and 6 weeks. Anti-RSV F antibodytitres (IgG, IgG1 and IgG2a) in these sera were determined byenzyme-linked immunosorbent assay (ELISA), as described in Example 4.The RSV-specific plaque reduction titres of these sera were determinedas previously described (ref. 44).

The anti-RSV F antibody responses in the sera of BALB/c mice that wereimmunized as outlined in Table 1 are summarized in FIG. 3. The animalsimmunized with SFV-RSVF RNA, live RSV, or RSV subunit preparation+alumall had high total anti-RSV F IgG antibody titres in their serum at both4 and 6 weeks (FIG. 3, panel A). However, the IgG1/IgG2a ratios differedmarkedly, as seen from FIG. 3, panels B and C. The sera from animalsthat were immunized with live RSV had an anti-F IgG1/IgG2a ratio ofapproximately 0.69 after 6 weeks. This value is in contrast to theanti-RSV F IgG1/IgG2a ratio obtained in mice after 6 weeks that wereprimed and boosted with the alum-adjuvanted RSV subunit preparation. Inthis case, the anti-RSV IgG1/IgG2a ratio was approximately 4.3. Theanti-RSV F IgG1/IgG2a ratios obtained in mice immunized with SFV-RSVFRNA after 6 weeks were 0.79. These results suggest that immunization ofmice with the SFV-RSVF RNA results in more of a Th-1 type responsesimilar to that obtained with live RS virus rather than the Th-2 typeresponse seen with the alum-adjuvanted RSV subunit preparation.

As shown in FIG. 4, the sera of mice that were primed and boosted withthe various RSV preparations as outlined in Table 1, all had significantlevels of RSV-specific neutralizing antibodies (groups 2, 3 and 5). Incontrast to the placebo control animals (groups 1 and 4), the lowerrespiratory tract of mice that were immunized with SFV-RSVF RNA, liveRSV, or the alum-adjuvanted RSV subunit preparation, were completelyprotected against live RS virus challenge, as seen in Table 2.

Immunization of mice with the SFV-RSVF RNA protected mice against liveRSV challenge. The protective ability of this SFV replicon wascomparable to that induced by inoculation with live RSV oralum—adjuvanted RSV subunits. The type of immune response generatedappeared to be more of a Th-1 like response similar to that elicited bylive RSV.

Example 4

This Example describes the determination of anti-RSV F antibody titres.

Nunc-MaxiSorp plate wells were coated overnight at room temperature with2.5 ng of immunoaffinity-purified RSV F protein diluted in 0.05Mcarbonate-bicarbonate buffer, pH 9.6. Wells were blocked fornon-specific binding by adding 0.1% BSA in PBS for 30 min. at roomtemperature, followed by two washes in a washing buffer of 0.1% BSA inPBS+0.1% Tween 20. Serial two or four-fold dilutions of mouse serum wasadded to the wells. After a one hour incubation at room temperature,plates were washed five times with washing buffer, and horseradishperoxidase (HRP) labeled conjugate was added at the appropriate optimaldilution in washing buffer. The total IgG assay used F(ab′)₂ goatantimouse IgG (H+L specific)-HRP from Jackson Immuno Research LaboratoryInc. (Baltimore Md., USA). Sheep anti-mouse IgGl -HRP from Serotec(Toronto, Ontario, Canada) was used in the IgG1 assay and goatanti-mouse IgG2a from Caltag Laboratories (San Francisco, Calif., USA)was used in the IgG2a assay. Following one hour incubation at roomtemperature, the plates were washed five times with washing buffer, andhydrogen peroxide (substrate) in the presence of tetramethylbenzidinewas added. The reaction was stopped by adding 2 M sulfuric acid. Thecolour was read in a Multiscan Titertek plate reader at an opticaldensity (OD) of 450 nm. The titre was taken as the reciprocal of thelast dilution at which the OD was approximately double. This OD must begreater that the negative control of the assay at the starting dilution.The pre-immune serum of each animal was used as the negative control.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, there are provided novel vectorscontaining DNA sequences encoding a paramyxovirus protein, particularlya RSV F protein, which can be linearized and transcribed to RNA for invivo administration to generate a protective immune response to diseasecaused by infection by paramyxovirus, particularly respiratory syncytialvirus. Modifications are possible within the scope of this invention.

TABLE 1 Immunization protocol ROUTE OF ROUTE OF INOCULA- INOCULA- GROUPPRIME TION BOOST TION 1 SFV-LacZ Intra- SFV-LacZ Intramuscular RNA¹muscular RNA¹ 2 SFV-RSVF Intra- SFV-RSVF Intramuscular RNA¹ muscularRNA¹ 3 Live RSV² Intranasal Live RSV² Intranasal 4 PBS + alum Intra-PBS + alum Intramuscular muscular 5 RSV subunits + Intra- RSV subunits +Intramuscular alum³ muscular alum³ Mice were inoculated with: ¹25 μg ofRNA was injected into each hind leg muscle in 50 μL of PBS ²2.5 × 10⁵pfu of mouse-adapted A2 virus ³1 μg of RSV subunit vaccine adsorbed toalum (1.5 mg/dose)

TABLE 2 Mean Virus Lung Titre ? Antigen Formulation (log₁₀ /g ± pro-GROUP Prime Boost s.d.) tection 1 SFV-LacZ SFV-LaCZ RNA 4.18 ± 0.06 0RNA 2 SFV-RSVF SFV-RSVF RNA ≦1.83 ± 0 100 RNA 3 Live RSV Live RSV ≦1.83± 0 100 4 PBS + alum PBS + alum 4.17 ± 0.17 0 5 RSV subunits + RSVsubunits + ≦1.83 ± 0 100 alum alum

Reference

1. McIntosh K. and Chanock R. M. in Fields B. N. and Knipe D. M. (eds).Virology. Raven Press, New York, 1990, pp.1045-1072.

2. Murphy B. R., Hall S. L., Kulkarni A. B., Crowe J. E., Collins P. L.,Connors M., Karron R. A. and Chanock R. M., Virus Res 32, 13-36, 1994.

3. Osterweil D. and Norman D., Am Geriat Soc 36, 659-662, 1990.

4. Agius G., Dindinand G., Biggar R. J., Peyre R., Vaillant V., RangerS., Poupet J. Y., Cisse M. F. and Casters M., J Med Virol 30, 117-127,1990.

5. Katz S. L. in New vaccine development establishing priorities Vol 1.National Academic Press, Washington, 1985, pp. 3974 09.

6. Wertz G. W. and Sullender W. M., Biotechnology 20, 151-176, 1992.

7. Fulginiti V. A., Eller J. J., Sieber O. F., Joyner J. W., MinamitaniM. and Meiklejohn G., Am i Epidemiol 89, 449-463, 1969.

8. B. Chin J., Magoffin R. L., Shearer I. A., Schieble J. H. andLennette E. H., Am J Epidemiol 89, 449-463, 1969.

9. Belshe R. B., Van Voris L. P. and Mufson M. A., J Infect Dis 145,311-319, 1982.

10. Kim R. M., Arrobio J. O., Pyles G., Brandt C. D., Camargo E.,Chanock R. M. and Parrott R. H., Pediatrics 48, 745-755, 1971.

11. Gruber C. and Levine S., J Gen Virol 64, 825-832, 1983.

12. Olmstead R. A., Elango N. and Prince G. A., Proc Natl Acad Sci USA83, 7462-7466, 1991.

13. Parrington M., Cockle S., Wyde P., Du R. -P., Snell E., Yan W.-Y.,Wang Q., Gisonni L., Sanhueza S., Ewasyshyn M. and Klein M., Virus Genes14, 65-74, 1997

14. Fulginiti, V. A., Eller, J. J., Sieber, O. F., Joyner, J. W.,Minamitani, M. and Meiklejohn, G. (1969) Am. J. Epidemiol. 89 (4),435-448.

15. Chin, J., Magoffin, R. L., Shearer, L. A., Schieble, J. H. andLennette, E. H. (1969) Am. J. Epidemiol. 89 (4), 449-463.

16. Jensen, K. E., Peeler, B. E. and Dulworth, W. G. (1962) J. Immunol.89, 216-226.

17. Murphy, B. R., Prince, G. A., Collins, P. L., Van Wyke -Coelingh,K., Olmsted, R. A., Spriggs, M. K., Parrott, R. H., Kim, H.-Y., Brandt,C. D. and Chanock, R. M. (1988) Vir. Res. 11, 1-15.

18. Hall, S. L., Sarris, C. M., Tierney, E. L., London, W. T., andMurphy, B. R. (1993) J. Infect. Dis. 167, 958-962.

19. Belshe, R. B., Karron, R. A., Newman, F. K., Anderson, E. L.,Nugent, S. L., Steinhoff, M., Clements, M. L., Wilson, M. H., Hall, S.L., Tierney, E. L. and Murphy, B. R. (1992) J. Clin. Microbiol. 30 (8),2064-2070.

20. Hall, S. L., Stokes, A., Tierney, E. L., London, W. T., Belshe, R.B., Newman, F. C. and Murphy, B. R. (1992) Vir. Res. 22, 173-184.

21. Van Wyke Coelingh, K. L., Winter, C. C., Tierney, E. L., London, W.T. and Murphy, B. R. (1988) J. Infect. Dis. 157 (4), 655-662.

22. Ray, R., Novak, M., Duncan, J. D., Matsuoka, Y. and Compans, R. W.(1993) J. Infect. Dis. 167, 752-755.

23. Ray, R., Brown, V. E. and Compans, R. W. (1985) J. Infect. Dis. 152(6), 1219-1230.

24. Ray, R. and Compans, R. W. (1987) J. Gen. Virol. 68, 409-418.

25. Ray, R., Glaze, B. J., Moldoveanu, Z. and Compans, R. W. (1988) J.Infect. Dis. 157 (4), 648-654.

26. Ray, R., Matsuoka, Y., Burnett, T. L., Glaze, B. J. and Compans, R.W. (1990) J. Infect. Dis. 162, 746-749.

27. Ray, R., Glaze, B. J. and Compans, R. W. (1988) J. Virol. 62 (3),783-787.

28. Ewasyshyn, M., Caplan, B., Bonneau A.-M., Scollard, N., Graham, S.,Usman, S. and Klein, M. (1992) Vaccine 10 (6), 412-420.

29. Ambrose, M. W., Wyde, P. R., Ewasyshyn, M., Bonneau, A.-M., Caplan,B., Meyer, H. L. and Klein, M. (1991) Vaccine 9, 505-511.

30. Kasel, J. A., Frank, A. L., Keitel, W. H., Taber, L. H., Glezen W.P. J. Virol. 1984; 52:828-32.

31. Lehman, D. J., Roof, L. L., Brideau, R. J., Aeed, P. A., Thomsen, D.R., Elhammer, A. P., Wathen, M. W. and Homa, F. L. (1993) J. Gen. Virol.74, 459-469.

32. Brideau, R. J., Oien, N. L., Lehman, D. J., Homa, F. L. and Wathen,M. W. (1993) J. Gen. Virol. 74, 471-477.

33. Ebata, S. N., Prevec, L., Graham, F. L. and Dimock, K. (1992) Vir.Res. 24, 21-33.

34. Hall, S. L., Murphy, B. R. and Van Wyke Coelingh, K. L. (1991)Vaccine 9, 659-667.

35. Strauss E. G. and Strauss J. H., in Schlesinger S. S. andSchlesinger M. i. (eds). The Togaviridae and Flaviviridae. Plenum Press,New York, 1986, pp. 35-90.

36. Liljestrom P. and Garoff H., Biotechnology 9, 1356-1361, 1991.

37. Zhou X., Berglund P., Rhodes G., Parker S. E., Jondal M. andLiljestrom P., Vaccine 12, 1510-1514, 1994.

38. Dalemans W., Delers A., Delmelle C., Denamur F., Meykens R.,Thiriart C., Veenstra S., Francotte M., Bruck C. and Cohen J., AnnalsNew York Academy of Sciences, 255-256, 1996.

39. Tang et al, Nature 1992, 356: 152-154.

40. Futh et al, Analytical Biochemistry, 1992, 205: 365-368.

41. Du, R. P. et al., Biotechnology 12, 813-818, 1994

42. Graham B. S., Perkins M. D., Wright P. F. and Karzon D. T., J. Mod.Virol. 26, 153-162, 1988.

43. Davis et al. Vaccine 12, 1503-1509, 1994.

44. Prince, G. A., et al. Am. J. Pathol. 93, 771-790, 1978.

7 1 1623 DNA respiratory syncytial virus 1 gatccgcgcg cgcgaattcggcacgagtaa caatggagtt gctaatcctc aaagcaaatg 60 caattaccac aatcctcactgcagtcacat tttgttttgc ttctggtcaa aacatcactg 120 aagaatttta tcaatcaacatgcagtgcag ttagcaaagg ctatcttagt gctctgagaa 180 ctggttggta taccagtgttataactatag aattaagtaa tatcaaggaa aataagtgta 240 atggaacaga tgctaaggtaaaattgataa aacaagaatt agataaatat aaaaatgctg 300 taacagaatt gcagttgctcatgcaaagca caccaccaac aaacaatcga gccagaagag 360 aactaccaag gtttatgaattatacactca acaatgccaa aaaaaccaat gtaacattaa 420 gcaagaaaag gaaaagaagatttcttggtt ttttgttagg tgttggatct gcaatcgcca 480 gtggcgttgc tgtatctaaggtcctgcacc tagaagggga agtgaacaag atcaaaagtg 540 ctctactatc cacaaacaaggctgtagtca gcttatcaaa tggagttagt gtcttaacca 600 gcaaagtgtt agacctcaaaaactatatag ataaacaatt gttacctatt gtgaacaagc 660 aaagctgcag catatcaaatatagaaactg tgatagagtt ccaacaaaag aacaacagac 720 tactagagat taccagggaatttagtgtta atgcaggtgt aactacacct gtaagcactt 780 acatgttaac taatagtgaattattgtcat taatcaatga tatgcctata acaaatgatc 840 agaaaaagtt aatgtccaacaatgttcaaa tagttagaca gcaaagttac tctatcatgt 900 ccataataaa agaggaagtcttagcatatg tagtacaatt accactatat ggtgttatag 960 atacaccctg ttggaaactacacacatccc ctctatgtac aaccaacaca aaagaagggt 1020 ccaacatctg tttaacaagaactgacagag gatggtactg tgacaatgca ggatcagtat 1080 ctttcttccc acaagctgaaacatgtaaag ttcaatcaaa tcgagtattt tgtgacacaa 1140 tgaacagttt aacattaccaagtgaaataa atctctgcaa tgttgacata ttcaacccca 1200 aatatgattg taaaattatgacttcaaaaa cagatgtaag cagctccgtt atcacatctc 1260 taggagccat tgtgtcatgctatggcaaaa ctaaatgtac agcatccaat aaaaatcgtg 1320 gaatcataaa gacattttctaacgggtgcg attatgtatc aaataaaggg atggacactg 1380 tgtctgtagg taacacattatattatgtaa ataagcaaga aggtaaaagt ctctatgtaa 1440 aaggtgaacc aataataaatttctatgacc cattagtatt cccctctgat gaatttgatg 1500 catcaatatc tcaagtcaacgagaagatta accagagcct agcatttatt cgtaaatccg 1560 atgaattatt acataatgtaaatgctggta aatccaccac aaatatcatg acttgataat 1620 gag 1623 2 527 PRTrespiratory syncytial virus 2 Met Glu Leu Leu Ile Leu Lys Ala Asn AlaIle Thr Thr Ile Leu Thr 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser GlyGln Asn Ile Thr Glu Glu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val SerLys Gly Tyr Leu Ser Ala Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val IleThr Ile Glu Leu Ser Asn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr AspAla Lys Val Lys Leu Ile Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys AsnAla Val Thr Glu Leu Gln Leu Leu 85 90 95 Met Gln Ser Thr Pro Pro Thr AsnAsn Arg Ala Arg Arg Glu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr LeuAsn Asn Ala Lys Lys Thr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg LysArg Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile AlaSer Gly Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160 Glu Gly GluVal Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala ValVal Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190 LeuAsp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215220 Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225230 235 240 Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn SerGlu 245 250 255 Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp GlnLys Lys 260 265 270 Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln SerTyr Ser Ile 275 280 285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr ValVal Gln Leu Pro 290 295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp LysLeu His Thr Ser Pro 305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu GlySer Asn Ile Cys Leu Thr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys AspAsn Ala Gly Ser Val Ser Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys LysVal Gln Ser Asn Arg Val Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu ThrLeu Pro Ser Glu Ile Asn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn ProLys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr 385 390 395 400 Asp Val SerSer Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr GlyLys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 LysThr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp 435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455460 Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465470 475 480 Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln ValAsn 485 490 495 Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser AspGlu Leu 500 505 510 Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn IleMet Thr 515 520 525 3 17 DNA respiratory syncytial virus 3 catgacttgataatgag 17 4 17 DNA respiratory syncytial virus 4 tgaactatta ctcctag 175 14 DNA respiratory syncytial virus 5 gatccgcgcg cgcg 14 6 14 DNArespiratory syncytial virus 6 gcgcgcgcgc ttaa 14 7 25 DNA respiratorysyncytial virus 7 tggttggtat accagtgtta taact 25 1 2

I claim:
 1. A vector, comprising: a first DNA sequence which iscomplementary to at least part of an alphavirus RNA genome and having acomplement of complete alphavirus RNA genome replication regions, and asecond DNA sequence which encodes an RSV F protein and lacks a Spelrestriction site and lacks a transmembrane anchor and cytoplasmic tailencoding region, said second DNA sequence having a nucleotide sequenceselected from the group consisting of SEQ ID No: 1 and a nucleotidesequence encoding SEQ ID No: 2, said second DNA sequence being insertedinto a region of said first DNA sequence which is non-essential forreplication thereof, said first and second DNA sequences being undertranscriptional control of a promoter.
 2. The vector of claim 1 whereinsaid alphavirus is a Semliki Forest virus.
 3. The vector of claim 2wherein said first DNA sequence is the Semliki Forest virus sequencecontained in plasmid pSFV1.
 4. The vector of claim 2 wherein saidpromoter is the SP6 promoter.
 5. The vector of claim 1 which is aplasmid vector having a unique restriction site permitting linearizationof the vector without cleaving the second DNA sequence.
 6. The vector ofclaim 5 wherein the unique restriction site is SpeI site.
 7. The vectorof claim 7 wherein the SpeI site is derived from plasmid pSFV1.
 8. Thevector of claim 1 is in a linearized form.
 9. An RNA transcript of thevector of claim
 1. 10. The RNA transcript of claim 9 which is derived bylinearization of a plasmid vector of claim 1 at a unique restrictionsite provided in the vector and permitting linearization of the vectorwithout cleaving the second DNA sequence and transcribing the linearizedvector.
 11. The RNA transcript of claim 9 wherein, in said vector, saidalphavirus is a Semliki Forest virus.
 12. The RNA transcript of claim 11wherein, in said vector, said first DNA sequence is the Semliki Forestvirus sequence contained in plasmid pSFV1.
 13. The RNA transcript ofclaim 11 wherein, in said vector, said promoter is the SP6 promoter. 14.An immunogenic composition for in vivo administration to a host for thegeneration in the host of antibodies to paramyxoviridae protein,comprising, as the active component thereof, an RNA transcript asclaimed in claim
 9. 15. The immunogenic composition of claim 14 for thegeneration of protective antibodies in the host.
 16. A method ofimmunizing a host against disease caused by infection withparamyxovirus, which comprises administering to said host an effectiveamount of an RNA transcript as claimed in claim
 9. 17. A mutant DNAsequence encoding an RSV F from which is absent an Spel restriction sitepresent in the non-mutant sequence, said mutant DNA sequence lacking atransmembrane anchor and cytoplasmic tail encoding region.
 18. Themutant DNA sequence of claim 17 wherein nucleotide 194 (T) of anon-mutant RSV F gene is mutated to a C to eliminate the Spel site inthe non-mutant RSV F gene.
 19. A mutant DNA molecule sequence lacking anSpeI site present in the non-mutant sequence and encoding a truncatedRSV F protein and having the DNA sequence shown in FIG. 2 (SEQ ID No:1)or having a DNA sequence encoding the amino acid sequence shown in FIG.2 (SEQ ID No: 2).
 20. A method of using a gene encoding a respiratorysyncytial virus (RSV) F protein to protect a host against infectioncaused by RSV, which comprises: isolating said gene, said gene lacking aSpel restriction site and encoding a RSV F protein that lacks atransmembrane anchor and cytoplasmic tail, said gene having a nucleotidesequence selected from the group consisting of SEQ ID No: 1 and anucleotide sequence encoding SEQ ID No: 2, operatively linking said geneto a DNA sequence which is complementary to at least part of analphavirus RNA gemone and having the complement of complete alphavirusRNA genome replication regions in a region of said DNA sequence which isnon-essential for replication to form a plasmid vector wherein said geneand DNA sequence are under transcriptional control of a promoter,linearizing the plasmid vector while maintaining said gene and DNAsequence under said transcriptional control of the promoter, forming anRNA transcript of said linearized vector, and introducing said RNAtranscript to a host.
 21. The method of claim 20 wherein saidlinearizing of the plasmid vector is effected by cleaving the plasmidvector at a unique restriction site therein at a location which permitsthe maintenance of the gene and DNA sequence under transcriptionalcontrol of the promoter.
 22. The method of claim 21 wherein the uniquerestriction site is an SpeI site.